Tissue type plasminogen activator (tPA) is a serine protease secreted in the neurovascular unit (NVU) by endothelial cells (Angles-Cano et al., 1985), neurons (Pecorino et al., 1991) and glial cells ((Siao and Tsirka, 2002); (Buisson et al., 1998)). Unlike other serine proteases, tPA is an unusually active zymogen, with full intrinsic activity and low zymogenicity (Loscalzo, 1988). In the vasculature, tPA promotes fibrinolysis via the conversion of the abundant and inactive fibrin-bound zymogen plasminogen into plasmin. In the brain parenchyma, tPA was reported to display critical functions such as the control of the neuronal migration, learning and memory processes notably through the control of the N-methyl-D-aspartate receptor (NMDAR) signalling ((Calabresi et al., 2000); (Nicole et al., 2001); (Su et al., 2008); (Seeds et al., 1999)).
At the time when tPA (clinically delivered as Actilyse® or Alteplase®) was approved by the Federal Food and Drug Administration for the acute treatment of ischemic stroke, experimental data favour the idea that beyond its beneficial vascular effects, tPA may have damaging properties in the cerebral parenchyma, including haemorrhagic transformations and neurotoxicity ((Fugate et al., 2010); (Yepes et al., 2009)). Indeed, beyond its ability to promote clot lysis, it is now well established, from both experimental models and clinical data, that tPA can activate metalloproteinases, growth factors, mediates neutrophils activation and thus promotes haemorrhagic transformations ((Suzuki et al., 2009); (Fredriksson et al., 2004); (Rosell et al., 2008)). Interestingly, intravenous tPA is also capable to cross both the intact and the injured blood brain barrier ((Harada et al., 2005); (Benchenane et al., 2005); (Benchenane et al., 2005)) and thus influence brain dysfunctions such as neurotoxicity ((Samson and Medcalf, 2006); (Benchenane et al., 2007); for review, (Yepes et al., 2009)).
Accordingly, in the NVU and together with endogenous parenchymal tPA, blood derived tPA interacts with several substrates in vitro and in vivo. Among its mechanisms of action, by interacting with the N-methyl-D-aspartate receptors (NMDAR) in neurons tPA is known to activate NMDAR-dependent signaling processes leading to an exacerbated neuronal death in conditions of oxygen and glucose deprivation, excitotoxicity or ischemia (Nicole et al., 2001) (Baron et al., 2010).
It is thus of major concern to identify tPA derivatives which would present a good or improved fibrinolytic activity, but without having the damaging properties in the cerebral parenchyma of the existing tPA, including exacerbated neuronal death.
It is also of major concern that said tPA derivatives have a reasonable intrinsic activity (which may be measured thanks to their amidolytic activity), so that vascular adverse effects are minimized.
The inventors have identified specific mutated tPA, which are efficient thrombolytics, which have a reasonable intrinsic activity, and which do not promote NMDAR-mediated neurotoxicity.